Chloride Channel Blocker Research Articles

Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia.

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l−1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l−1) to a hypotonic solution (290 mOsm l−1), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4′-diisothiocyanatostilbene-2,2′- isulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.

Inclusion of an Anthracene-based Fluorophore within Molecular Containers: A Comparative Study of the Cucurbituril and Cyclodextrin Host Families.

In this paper, the binding interaction of a promising chloride channel blocker, 9-methyl anthroate (9-MA), with two different classes of molecular containers, β-cyclodextrins (β-CD and methyl-β-CD) and cucurbit[7]uril, having comparable cavity dimensions, has been thoroughly demonstrated via inspection of the modulation of the excited-state properties of the emissive molecule. Spectral data suggest that CB7 encapsulates the probe more efficiently in a 1:2 fashion, whereas the efficacies of β-CDs are relatively less and the corresponding stoichiometry is 1:1. Interestingly, despite being thermodynamically much more favorable than the probe-β-CD complexation equilibria, the fraction of probe-CB7 complex formed is appreciably smaller with respect to that of probe-β-CD complexes. This apparent inconsistency has been addressed via the proposition that since the formation of a 1:2 complex is entropically disadvantageous, it is anticipated that the activation barrier of the corresponding reaction is reasonably high, and thus only a small fraction of the reactants are able to surpass the energy barrier to form the products. This proposition has been thoroughly corroborated by fluorescence lifetime measurements at different temperatures.

The volatile constituent analysis of Digitalis nervosa Steud. & Hochst. ex Benth. from Iran

The volatile constituents of the leaves of Digitalis nervosa Steud. & Hochst. ex Benth. growing wild in Iran was reported for the first time. The essential oil of the leaves of this plant was examined by gas chromatography using a flame-ionization detector and gas chromatography coupled with mass spectrometry. Thirty-four components were identified constituting approximately 96.5% of the essential oil of D. nervosa. The essential oil of D. nervosa is rich in palmitic acid (20.5%), trans-pinocamphone (16.0%), phytol (6.5%), caryophyllen oxide (5.9%), hexahydrofarnesyl acetone (4.6%), 3-ethyl-4-methyl-1-pentanol (4.4%), oleic acid (3.7%), spathulenol (3.6%), di(2-methylpropyl) phthalate (2.7%), cedrol (2.3%), and farnesol (2.1%). The results obtained showed that the essential oil of D. nervosa contains ten oxygenated monoterpenes (25.7%), seven oxygenated sesquiterpenes (21.1%), one oxygenated diterpene (6.5%), and fourteen nonterpene (41.4%) compounds. The essential oil from the D. nervosa leaves has relatively high content of the oxygenated monoterpenes, therefore a significant antibacterial role for this essential oil is expectable. Because of the presence of trans-pinocamphone, which can act as a γ-aminobutyric acid (GABA)-gated chloride channel blocker, the essential oil of D. nervosa can be neurotoxic.

GABAA receptor cysteinyl mutants and the ginkgo terpenoid lactones bilobalide and ginkgolides

The terpenoid lactones from Ginkgo biloba, bilobalide and ginkgolides, have been shown to act as negative modulators at α1β2γ2L GABAA receptors. They have structural features similar to those of the chloride channel blocker picrotoxinin. Unlike picrotoxinin, however they are not known to produce convulsant effects. Using two-electrode voltage clamp electrophysiology, this study compared the effect of mutation of 2′, 6′ and 15′ pore facing M2 domain residues to cysteine on the action of picrotoxinin, bilobalide and ginkgolides at α1β2γ2L GABAA receptors expressed in Xenopus oocytes. Picrotoxinin was affected by mutation differently from the ginkgo terpenoid lactones. Although some of these compounds were affected by the mutation at same position and/or subunit, the changes in their potency were found to be dissimilar. The results suggest that the intracellular pore binding site for picrotoxinin, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C is comprised of 2′β-6′β6′γ, 2′α2′β-6′α6′β, 2′α2′β2′γ-6′β6′γ, 2′α, 2′β2′γ-6′β and 2′α2′β, respectively. Unlike bilobalide and ginkgolides, the inhibitory action of picrotoxinin was not affected by mutations at 15′ position. It is proposed that 15′α15′β, 15′β, 15′α15′β and 15′α15′β15′γ forms an extracellular pore binding site for bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, respectively. The lack of convulsant effects of bilobalide, and ginkgolide A and B may be associated in part with their different binding locations within the chloride channel.

Pharmacological induction of skin pigmentation unveils the neuroendocrine circuit regulated by light.

Light-regulated skin colour change is an important physiological process in invertebrates and lower vertebrates, and includes daily circadian variation and camouflage (i.e. background adaptation). The photoactivation of melanopsin-expressing retinal ganglion cells (mRGCs) in the eye initiates an uncharacterized neuroendocrine circuit that regulates melanin dispersion/aggregation through the secretion of alpha-melanocyte-stimulating hormone (α-MSH). We developed experimental models of normal or enucleated Xenopus embryos, as well as in situ cultures of skin of isolated dorsal head and tails, to analyse pharmacological induction of skin pigmentation and α-MSH synthesis. Both processes are triggered by a melanopsin inhibitor, AA92593, as well as chloride channel modulators. The AA9253 effect is eye-dependent, while functional data in vivo point to GABAA receptors expressed on pituitary melanotrope cells as the chloride channel blocker target. Based on the pharmacological data, we suggest a neuroendocrine circuit linking mRGCs with α-MSH secretion, which is used normally during background adaptation.

Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker.

Background and PurposeCalcium‐activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene‐9‐carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined.Experimental ApproachPatch‐clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels.Key ResultsIn the presence of high intracellular Ca2+, A9C inhibited TMEM16A currents in a voltage‐dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca2+ concentrations, was also voltage‐dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open‐channel block mechanism. Activation was due to a dramatic leftward shift in the steady‐state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl−, suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects.Conclusions and ImplicationsA9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels.

Double Blockade of Glioma Cell Proliferation and Migration by Temozolomide Conjugated with NPPB, a Chloride Channel Blocker.

Glioblastoma is the most common and aggressive primary malignant brain tumor. Temozolomide (TMZ), a chemotherapeutic agent combined with radiation therapy, is used as a standard treatment. The infiltrative nature of glioblastoma, however, interrupts effective treatment with TMZ and increases the tendency to relapse. Voltage-gated chloride channels have been identified as crucial regulators of glioma cell migration and invasion by mediating cell shape and volume change. Accordingly, chloride current inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a chloride channel blocker, suppresses cell movement by diminishing the osmotic cell volume regulation. In this study, we developed a novel compound, TMZ conjugated with NPPB (TMZ-NPPB), as a potential anticancer drug. TMZ-NPPB blocked chloride currents in U373MG, a severely invasive human glioma cell line, and suppressed migration and invasion of U373MG cells. Moreover, TMZ-NPPB exhibited DNA modification activity similar to that of TMZ, and surprisingly showed remarkably enhanced cytotoxicity relative to TMZ by inducing apoptotic cell death via DNA damage. These findings indicate that TMZ-NPPB has a dual function in blocking both proliferation and migration of human glioma cells, thereby suggesting its potential to overcome challenges in current glioblastoma therapy.

Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma.

Background: Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma.Methods: Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment).Results: Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects.Conclusion: The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone.

ClC-3 is Involved in NPPB-Induced Apoptosis in DU145 Prostate Cancer Cells

NPPB3-(4-5-nitro-2-(3-phenylpropylamino) benzoic acid, as one of the most commonly used chloride channel blockers, is very essential for chloride channel studies. However, the role of NPPB in mediating apoptosis is uncertain. The purpose of this study was to investigate the apoptosis mechanism of NPPB in human cancer DU145 cell lines. Cell viability was examined by MTT assay. JC-1 fluorescent probe was used for detecting l membrane potential. Cell apoptosis was determined by flow method (FCM). The expression of apoptosis-related protein Caspase-3, Cleaved caspase-3, PARP, Cleaved PARP, Bcl-2, Mcl-1, Bcl-xl, Bcl-w and Bax was detected by western blotting. Cell proliferation rate was remarkably inhibited 2 h after treating with NPPB(50,100 μmol/L) (p<0.01); Flow cytometry method analysis showed that the apoptosis rate in each treated group were significantly higher, especially for the promotion of early apoptosis rate effect was significantly (P<0.05); Images of JC-1 showed NPPB led to the decrease of intracellular mitochondrial membrane potential; Western blot results showed that caspase-3/PARP signaling pathway was activated, while the expression of cleaved caspase-3, cleaved PARP was significantly increased (P<0.01).PARP, as substrate of caspase-3, was significantly decreased (P<0.05).Bcl-2, Bcl-xl compared with the control group had no significant difference, and anti-apoptotic protein Mcl-1 (P<0.05) and Bcl-w (P<0.01) decreased significantly, pro-apoptotic protein Bax (P<0.01) were obviously upregulated. Our findings indicated that NPPB could increase apoptosis in DU145 cell lines via mitochondria-related apoptotic pathway, and following results presented that ClC-3 Chloride Channels involved in the process of NPPB induced DU145 cells apoptosis.

ClC-3 Expression and Its Association with Hyperglycemia Induced HT22 Hippocampal Neuronal Cell Apoptosis.

Although apoptosis plays an important role in the development of Diabetic Encephalopathy (DE), the underlying molecular mechanisms remain unclear. With respect to this, the present work aims to study the variation in chloride/proton exchanger ClC-3 expression and its association with HT22 hippocampal neuronal apoptosis under hyperglycemic condition in vitro. The cells were stimulated with added 0, 5, or 25 mM glucose or mannitol for up to 72 hours before assessing the rate of ClC-3 expression, cell viability, and apoptosis. In a consecutive experiment, cells received chloride channel blocker in addition to glucose. The rate of cellular death/apoptosis and viability was measured using Flow Cytometry and MTT assay, respectively. Changes in ClC-3 expression were assessed using immunofluorescence staining and western blot analysis. The results revealed a significant increase in cellular apoptosis and reduction in viability, associated with increased ClC-3 expression in high glucose group. Osmolarity had no role to play. Addition of chloride channel blocker completely abolished this effect. Thus we conclude that, with its increased expression, ClC-3 plays a major role in hyperglycemia induced hippocampal neuronal apoptosis. To strengthen our understanding of this aforesaid association, we conducted an extensive literature search which is presented in this paper.

Electrical stimulation of adipose-derived mesenchymal stem cells in conductive scaffolds and the roles of voltage-gated ion channels

Since electrical stimulation (ES) can significantly accelerate bone healing, a conductive scaffold that can deliver ES locally at the defect site is desirable for bone defect therapy. Herein, an electrically conductive scaffold was prepared via incorporation of polypyrrole (PPY) in a polycaprolactone (PCL) template scaffold. In vitro tests with mouse osteoblasts indicate that the PPY/PCL scaffold has good biocompatibility, and is suitable for use as an ES substrate. When human adipose-derived mesenchymal stem cells (AD-MSCs) were cultured in the PPY/PCL scaffold and subjected to 200μA of direct current for 4h per day for 21days, the amount of calcium deposited was 100% higher than that without ES. When these cells were subjected to ES together with blockers of voltage-gated calcium (Ca2+v), sodium (Na+v), potassium (K+v), or chloride (Cl−v) channels, the ES-induced enhancement of AD-MSCs’ functions was reduced with Na+v, K+v, or Cl−v blockers and completely nullified with Ca2+v blocker. These results indicate that ion fluxes through these channels activated by ES induce different cascades of reactions in the cells, which subsequently regulate AD-MSCs’ functions, and Ca2+v plays a more critical role than the other three channels. Our results further the current understanding of the mechanisms by which ES regulates stem cells’ behavior, and also showed that the conductive PPY/PCL scaffold with application of ES has good potential in bone defect therapy. Statement of SignificanceIn this work, an electrically conductive and biocompatible scaffold was prepared by incorporating polypyrrole in a polycaprolactone template scaffold. Application of 200μA direct current for 4h per day to human adipose derived-mesenchymal stem cells cultured on this scaffold promoted migration of these cells into the inner region of the scaffold and enhanced their osteogenic differentiation. The roles of voltage-gated ion channels (Ca2+v, Na+v, K+v and Cl−v) in osteogenic differentiation stimulated by the electric current were investigated. The results from these experiments further the current understanding of the mechanisms by which electrical stimulation regulates stem cells’ behavior, and also show that the polypyrrole–polycaprolactone scaffold with application of electrical stimulation has good potential in bone defect therapy.

Ethanol Promotes Cell Migration via Activation of Chloride Channels in Nasopharyngeal Carcinoma Cells.

Excessive alcohol consumption has been identified as a significant risk factor for cancer development. Chloride channels have been proved previously by us and others to be involved in cancer cell migration. However, it is unknown whether chloride channels are associated with the effects of ethanol (EtOH) on cancer cell activities. The effects of EtOH on migration were detected by the wound healing assay in the nasopharyngeal carcinoma cells (CNE-2Z) and the normal nasopharyngeal epithelial cells (NP69-SV40T). The whole-cell patch clamp technique was used to record the EtOH-induced chloride current. The characteristics of the current were studied by anion substitution, hypertonic challenges, and channel blockers. EtOH promoted the migration of cancerous CNE-2Z cells, but could hardly affect the migration of normal NP69-SV40T cells. The EtOH-induced migration could be inhibited by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. The exposure of CNE-2Z cells to EtOH activated a chloride current, with the ion selectivity of I(-) >Br(-) > Cl(-) >gluconate, demonstrated by ion substitution experiments. EtOH could still activate a similar chloride current in the absence of Ca(2+) in the medium. The current could be inhibited by the hypertonicity-induced cell shrinkage and the channel blockers NPPB and tamoxifen. EtOH could also activate a chloride current in normal NP69-SV40T cells, with the properties similar to those in CNE-2Z cells, but the current density was much smaller than that recorded in cancerous CNE-2Z cells. It has been demonstrated in this study that EtOH can activate chloride channels and promote cell migration in cancerous cells, but can hardly affect the activities in normal cells. The data suggest for the first time that EtOH may promote cell migration via activation of chloride channels; long-term exposure to EtOH may increase the incident of tumor metastasis.

4,4'-Diisothiocyanostilbene-2,2'-disulfonic Acid (DIDS) Ameliorates Ischemia-Hypoxia-Induced White Matter Damage in Neonatal Rats through Inhibition of the Voltage-Gated Chloride Channel ClC-2.

Chronic cerebral hypoperfusion is believed to cause white matter lesions (WMLs), leading to cognitive impairment. Previous studies have shown that inflammation and apoptosis of oligodendrocytes (OLs) are involved in the pathogenesis of WMLs, but effective treatments have not been studied. In this study, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a chloride (Cl−) channel blocker, was injected into chronic cerebral ischemia-hypoxia rat models at different time points. Our results showed that DIDS significantly reduced the elevated mRNA levels and protein expression of chloride channel 2 (ClC-2) in neonatal rats induced by ischemia-hypoxia. Meanwhile, DIDS application significantly decreased the concentrations of reactive oxygen species (ROS); and the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha TNF-α in neonatal rats with hypoxic-ischemic damage. Myelin staining was weaker in neonatal rats with hypoxic-ischemic damage compared to normal controls in corpus callosum and other white matter, which was ameliorated by DIDS. Furthermore, the elevated number of caspase-3 and neural/glial antigen 2 (NG-2) double-labeled positive cells was attenuated by DIDS after ischemia anoxic injury. Administration of DIDS soon after injury alleviated damage to OLs much more effectively in white matter. In conclusion, our study suggests that early application of DIDS after ischemia-hypoxia injury may partially protect developing OLs.

Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na+ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na+. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.

Interaction of triblock co-polymer micelles with phospholipid-bilayer: a spectroscopic investigation using a potential chloride channel blocker.

Interaction of a potential chloride channel blocker, 9-methyl anthroate (9-MA), has been studied with zwitterionic l-α-phosphatidylcholine (egg-PC) lipid vesicles, which ascertains the utility of the drug as an efficient molecular reporter for probing the microheterogeneous environment of lipid-bilayers. The effect of a non-ionic triblock co-polymer P123 on the stability of these drug-bound lipid-bilayers has also been investigated by means of steady state and time-resolved spectroscopic techniques exploiting the fluorescence properties of the drug. Experimental results reveal that the addition of P123 to the drug-bound lipid results in a preferential complexation of the drug with the Pluronic leaving the lipid vesicles aside, which has been attributed to a substantially stronger binding interaction of the drug with P123 than that with egg-PC. The result is of potential interest from a medical perspective owing to the context of excess drug desorption from bio-membranes.

Spectroscopic and viscometric elucidation of the interaction between a potential chloride channel blocker and calf-thymus DNA: the effect of medium ionic strength on the binding mode.

The present study demonstrates a detailed characterization of the binding interaction of a potential chloride channel blocker 9-methyl anthroate (9-MA) with calf-thymus DNA. The modulated photophysical properties of the emissive molecule within the microheterogeneous bio-assembly have been spectroscopically exploited to monitor the drug-DNA binding interaction. Experimental results based on fluorescence and absorption spectroscopy aided with DNA-melting, viscometric and circular dichroism studies unambiguously establish the binding mode between the drug and DNA to be principally intercalative. Concomitantly, a discernible dependence of the mode of binding between the concerned moieties on the ionic strength of the medium is noteworthy. A dip-and-rise characteristic of the rotational relaxation profile of the drug within the DNA environment has been argued to be originating from a substantial difference in the lifetime as well as amplitude of the free and DNA bound drug molecule. In view of the prospective biological applications of the drug, the issue of facile dissociation of the intercalated drug from the DNA helix via a simple detergent-sequestration technique has also been unveiled. The utility of the present work resides in exploring the potential applicability of the fluorescence properties of 9-MA for studying its interactions with other relevant biological or biomimicking targets.

Volume-sensitive chloride channels are involved in cisplatin treatment of osteosarcoma

Chemotherapy is the most common therapeutic strategy used to treat osteosarcoma. The present study aimed to investigate the effects of functionally activated chloride channels on cisplatin-induced apoptosis of MG-63 human osteosarcoma cells. An MTT assay and flow cytometry were used to detect proliferation and apoptosis of the cells, respectively. Live cell imaging was used to detect volume changes in response to treatment with cisplatin and/or chloride channel blockers. The effects of these treatments on chloride currents were also assayed using the patch-clamp technique. The results of the present study indicate that chloride channel blockers may suppress cisplatin-induced apoptosis. The MG-63 cells cultured with cisplatin demonstrated an apoptotic volume decrease, as well as suppression of cell proliferation; which were reversed by co-treatment with chloride channel blockers. These results suggest that cisplatin may activate chloride channels, and that channel activation is an early signal in the pathways that lead to cisplatin-induced apoptosis and inhibition of proliferation in MG-63 cells. In conclusion, these results indicate that chloride channels have an important role in cisplatin treatment of osteosarcoma.

Binding of a potential chloride channel blocker with an anionic surfactant and subsequent release in solution: effect of a hydrotropic solute in the post-micellar region

The present work demonstrates a thorough description of the differential interactions of a potential chloride channel blocker, 9-methyl anthroate (9-MA), with a charged surfactant, sodium dodecyl sulfate (SDS), in the pre- and post-micellar regime along with the consequences of the addition of a hydrotropic salt in the post-micellar region. The modulated photophysical properties of the emissive probe within the biomimetic environment have been spectroscopically monitored to gain an insight into the binding interaction. The moderate strength of the binding phenomenon as revealed by Benesi–Hildebrand analysis, as well as the hindrance exerted by the binding process towards the aggregation of the surfactants, paves the way to rationalize the observation of the exclusion of the drug to the bulk after micelle formation to be an outcome of a free energy advantage in the thermodynamically favorable micellization process. A substantial increase in the bulk viscosity of the medium in the presence of the hydrotrope in the post-micellar region coupled with a non-covalent binding between the alkane chains of the salt and the hydrophobic drug has also been unveiled from steady state and time-resolved fluorimetric perspectives.

Rapid and slow chemical synaptic interactions of cholinergic projection neurons and GABAergic local interneurons in the insect antennal lobe.

The antennal lobe (AL) of insects constitutes the first synaptic relay and processing center of olfactory information, received from olfactory sensory neurons located on the antennae. Complex synaptic connectivity between olfactory neurons of the AL ultimately determines the spatial and temporal tuning profile of (output) projection neurons to odors. Here we used paired whole-cell patch-clamp recordings in the cockroach Periplaneta americana to characterize synaptic interactions between cholinergic uniglomerular projection neurons (uPNs) and GABAergic local interneurons (LNs), both of which are key components of the insect olfactory system. We found rapid, strong excitatory synaptic connections between uPNs and LNs. This rapid excitatory transmission was blocked by the nicotinic acetylcholine receptor blocker mecamylamine. IPSPs, elicited by synaptic input from a presynaptic LN, were recorded in both uPNs and LNs. IPSPs were composed of both slow, sustained components and fast, transient components which were coincident with presynaptic action potentials. The fast IPSPs were blocked by the GABAA receptor chloride channel blocker picrotoxin, whereas the slow sustained IPSPs were blocked by the GABAB receptor blocker CGP-54626. This is the first study to directly show the predicted dual fast- and slow-inhibitory action of LNs, which was predicted to be key in shaping complex odor responses in the AL of insects. We also provide the first direct characterization of rapid postsynaptic potentials coincident with presynaptic spikes between olfactory processing neurons in the AL.